Compositions of Near IR Closed Chain, Sulfo-Cyanine Dyes and Prostate Specific Membrane Antigen Ligands

ABSTRACT

Compositions and methods for visualizing tissue under illumination with fusion compounds of near-infrared radiation using near IR, closed chain, sulfo-cyanine dyes and prostate specific membrane antigen ligands are provided.

CROSS REFERENCE TO RELATED APPLICATION DATA

The present application is a Continuation of U.S. patent applicationSer. No. 16/593,207 filed Oct. 4, 2019; which is a Continuation of U.S.patent application Ser. No. 15/261,357 filed Sep. 9, 2016; which claimsthe benefit of U.S. Provisional Appln No. 62/324,097 filed Apr. 18,2016; the full disclosures which are incorporated herein by reference intheir entirety for all purposes.

BACKGROUND OF THE INVENTION

Minimally invasive medical techniques are intended to reduce the amountof extraneous tissue that is damaged during diagnostic or surgicalprocedures, thereby reducing patient recovery time, discomfort, anddeleterious side effects. While millions of “open” or traditionalsurgeries are performed each year in the United States; many of thesesurgeries can potentially be performed in a minimally invasive manner.One effect of minimally invasive surgery, for example, is reducedpost-operative recovery time and related hospital stay. Because theaverage hospital stay for a standard surgery is typically significantlylonger than the average stay for an analogous minimally invasivesurgery, increased use of minimally invasive techniques could savemillions of dollars in hospital costs each year. While many of thesurgeries performed in the United States could potentially be performedin a minimally invasive manner, only a portion currently employ thesetechniques due to instrument limitations, method limitations, and theadditional surgical training involved in mastering the techniques.

Minimally invasive tele-surgical systems are being developed to increasea surgeon's dexterity as well as to allow a surgeon to operate on apatient from a remote location. Telesurgery is a general term forsurgical systems where the surgeon uses some form of remote control,e.g., a servomechanism, or the like, to manipulate surgical instrumentmovements rather than directly holding and moving the instruments byhand. In such a telesurgery system, the surgeon is provided with animage of the surgical site at the remote location. While viewing thesurgical site on a suitable viewer or display, the surgeon performs thesurgical procedures on the patient by manipulating master control inputdevices, which in turn control the motion of instruments. These inputdevices can move the working ends of the surgical instruments withsufficient dexterity to perform quite intricate surgical tasks.

Minimally invasive medical techniques, including tele-surgical systemscan be further aided by improving visualization of the tissue where theprocedure is to be carried out. One way to improve visualization oftissue is through the use of dyes capable of targeted visualization oftissue, allowing a surgeon to either remove or spare said tissue. Thus,there is a need for novel, fusion compositions of near infrared (IR),closed chain, sulfo-cyanine dyes and prostate specific membrane antigen(PSMA) ligands as well as for novel methods of visualizing tissue underillumination with near-infrared radiation. Surprisingly, the presentinvention meets these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention generally provides novel, compositions and methodsfor visualizing tissue under illumination with fusion compoundscomprising near-infrared radiation using near IR, closed chain,sulfo-cyanine dyes and prostate specific membrane antigen ligands.

In a first embodiment, the present invention provides a compositioncomprising a compound having the formula:

wherein,

-   -   R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are each independently hydrogen,        C₁₋₄ alkyl, substituted C₁₋₄ alkyl, or —CO₂T;    -   X is a single bond, —O—, or —S—;    -   subscripts a, b, c, d, e, f, g, and h are each independently an        integer from 1 to 6, and    -   T is each independently a metal ion, H, or a negative charge;    -   Z is each independently H, C₁₋₆ substituted or unsubstituted        alkyl, C₆₋₁₂ substituted or unsubstituted aryl, or a C₆₋₁₂        substituted or unsubstituted heteroaryl; and

-   wherein, the composition is adapted for visualization of tissue    under illumination with near-infrared radiation.

In some cases, the composition has the formula:

In some cases, the composition has the formula:

In some cases, the composition is a pharmaceutical compositioncomprising a unit dosage form of a compound of formula I, II, or IIIwherein, the composition is adapted for administration to a patient; andwherein, the unit dosage form of the compound delivers to the patient anamount between 0.01 and 8 mg/kg. In some cases, the composition unitdosage form delivers to the patient the amount of 0.01, 0.05, 0.10,0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80,0.90, 1, 2, 4, 6, or 8 mg/kg. In some cases, the composition is dry anda single dose form.

In some embodiments, the composition is lyophilized in a sterilecontainer. In some cases, the composition is contained within a sterilecontainer, where the container has a machine detectable identifier thatis readable by a medical device.

In some embodiments, the composition further comprises combination withpharmaceutically acceptable excipients in an oral dosage form. In someembodiments, the composition further comprises combination withpharmaceutically acceptable carriers in an injectable dosage form. Insome embodiments, the composition further comprises combination withpharmaceutically acceptable excipients in a dosage form for directdelivery to a surgical site.

In some embodiments, the present invention provides a use of thecomposition adapted for administration to a patient to obtainvisualization of tissue expressing PSMA under illumination withnear-infrared radiation wherein the unit dosage form delivers to thepatient an amount is between 0.01 and 8 mg/kg. In some cases, the use isadapted for administration to a human patient to obtain visualization ofhuman tissue under illumination with near-infrared radiation wherein theunit dosage form delivers to the patient an amount between 0.01 and 8mg/kg.

In some embodiments, the present invention provides a method forvisualization of tissue expressing PSMA, the method comprising,administering to a patient a composition of a compound of formula I, II,or III and described herein. In some embodiments, the method comprises,administering to a patient a composition of a compound having theformula:

wherein,

-   -   R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are each independently hydrogen,        C₁₋₄ alkyl, substituted C₁₋₄ alkyl, or —CO₂T;    -   X is a single bond, —O—, or —S—;    -   subscripts a, b, c, d, e, f, g, and h are each independently an        integer from 1 to 6;    -   T is each independently a metal ion, H, or a negative charge; Z        is each independently H, C₁₋₆ substituted or unsubstituted        alkyl, C₆₋₁₂ substituted or unsubstituted aryl, or a C₆₋₁₂        substituted or unsubstituted heteroaryl; and where the compound        is administered in an amount sufficient for imaging tissue under        illumination with near-infrared radiation;        imaging the tissue under illumination with near-infrared        radiation; and obtaining at least one image of tissue from the        patient using the composition.

In some embodiments, the method administers to a patient apharmaceutical composition comprising a unit dosage form of a compoundof formula I wherein, the composition is sterile, non-toxic, and adaptedfor administration to a patient; and wherein, the unit dosage form ofthe compound delivers to the patient an amount between 0.01 and 8 mg/kg.In some cases, the method further comprises obtaining the image duringadministration, after administration, or both during and afteradministration of the composition. In some cases, the method furthercomprises intravenously injecting a composition of formula I into apatient. In some cases, the composition is injected into a circulatorysystem.

In some embodiments, the method further comprises visualizing a patientarea on which surgery is or will be performed, or for viewing a patientarea otherwise being examined by a medical professional. In some cases,the method further comprises performing a surgical procedure on thepatient areas based on the visualization of the surgical area. In somecases, the method further comprises viewing a patient area on which anophthalmic, arthroscopic, laparoscopic, cardiothoracic, muscular, orneurological procedure is or will be performed. In some cases, themethod further comprises obtaining ex vivo images of at least a portionof the patient.

In some embodiments, the tissue being visualized is tumor tissue. Insome cases, the tissue being visualized is dysplastic or canceroustissue. In some cases, the tissue being visualized is prostate tissue.In some cases, the tissue being visualized is prostate tumor tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows four fluorescence images. Each of the four fluorescenceimages show a similar tumor tissue treated with different fusioncompounds comprising different dyes and targeting ligands. The treatedtumor tissue in each of the four images was exposed to narrow band, nearIR excitation light. The narrow band, near IR excitation light includedlight wavelengths expected to correspond to the excitation maximawavelength for each of the fluorophores associated with each of thefusion compounds.

-   -   FIG. 1A shows tumor tissue treated with the fusion compound        having the composition DyLight800+PSMA-targeting ligand.    -   FIG. 1B shows tumor tissue treated with a fusion compound having        the composition ZW800+PSMA-targeting ligand. ZW800 is ZW800-1        manufactured by the FLARE Foundation or Curadel.    -   FIG. 1C shows tumor tissue treated with a fusion compound having        the composition IRDye800 CW+PSMA-targeting ligand. IRDye800CW is        manufactured by Li-Cor.    -   FIG. 1D shows tumor tissue treated with a fusion compound having        the composition Indocyanine green (ICG)+PSMA-targeting ligand.

FIG. 2 shows a bar graph plotting the average signal intensity detectedby an imaging sensor when each of the images was captured. The“I”-shaped bar at the top of each bar graph corresponds to the signalintensity standard deviation across the array of pixels making up theimage sensor.

-   -   The bar labeled Dylight corresponds to        DyLight800CW+PSMA-targeting ligand.    -   The bar labeled IRDye800 corresponds to IRDye800+PSMA-targeting        ligand.    -   The bar labeled ZW800 corresponds to ZW800+PSMA-targeting        ligand.    -   The bar labeled ICG corresponds to ICG+PSMA-targeting ligand.    -   The bar labeled “Control” corresponds to tumor tissue that was        not treated with any type of fluorophore-containing fusion        compound. The average signal intensity corresponding to the        Control is higher than the average signal intensities        corresponding to ZW800 and to ICG due to intrinsic noise of the        fluorescence imaging equipment used.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides novel, compositions and methods forvisualizing tissue under illumination with near-infrared radiation usingfusion compounds comprising near IR, closed chain, sulfo-cyanine dyesand prostate specific membrane antigen (PSMA) ligands. Surprisingly, thefusion compounds of present invention demonstrated significantlyincreased fluorescence compared other combinations of dyes to PSMAligands.

II. Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts.

The term “metal ion” as used herein refers to elements of the periodictable that are metallic and that are positively charged as a result ofhaving fewer electrons in the valence shell than is present for theneutral metallic element. Metals that are useful in the presentinvention include metals capable of forming pharmaceutically acceptablecompositions. Useful metals include Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr,and Ba. One of skill in the art will appreciate that the metalsdescribed above can each adopt several different oxidation states. Insome instances, the most stable oxidation state is formed, but otheroxidation states are useful in the present invention.

The term “alkyl” as used herein refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. For example, C₁-C₆ alkyl includes, but is not limited to,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include,but are not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl caninclude any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. Thealkyl group is typically monovalent, but can be divalent, such as whenthe alkyl group links two moieties together.

The term “cycloalkyl” as used herein refers to a saturated or partiallyunsaturated, monocyclic, fused bicyclic or bridged polycyclic ringassembly containing from 3 to 12 ring atoms, or the number of atomsindicated monocyclic rings include, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic andpolycyclic rings include, for example, norbornane, decahydronaphthaleneand adamantane. For example, C₃₋₈cycloalkyl includes cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

The term “haloalkyl” as used herein refers to alkyl as defined abovewhere some or all of the hydrogen atoms are substituted with halogenatoms. Halogen (halo) preferably represents chloro or fluoro, but mayalso be bromo or iodo. For example, haloalkyl includes trifluoromethyl,flouromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term “perfluoro”defines a compound or radical which has at least two available hydrogenssubstituted with fluorine. For example, perfluorophenyl refers to1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to1,1,1-trifluoromethyl, and perfluoromethoxy refers to1,1,1-trifluoromethoxy.

As used herein, the term “halogen” refers to fluorine, chlorine, bromineand iodine.

The term “aryl” as used herein refers to a monocyclic or fused bicyclic,tricyclic or greater, aromatic ring assembly containing 6 to 16 ringcarbon atoms. For example, aryl may be phenyl, benzyl or naphthyl,preferably phenyl. “Arylene” means a divalent radical derived from anaryl group. Aryl groups can be mono-, di- or tri-substituted by one, twoor three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen,cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy andoxy-C₂-C₃-alkylene; all of which are optionally further substituted, forinstance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or2-phenanthrenyl. Alkylenedioxy is a divalent substitute attached to twoadjacent carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy.Oxy-C₂-C₃-alkylene is also a divalent substituent attached to twoadjacent carbon atoms of phenyl, e.g. oxyethylene or oxypropylene. Anexample for oxy-C₂-C₃-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

The term “heteroaryl” as used herein refers to a monocyclic or fusedbicyclic or tricyclic aromatic ring assembly containing 5 to 16 ringatoms, where from 1 to 4 of the ring atoms are a heteroatom each N, O orS. For example, heteroaryl includes pyridyl, indolyl, indazolyl,quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl,furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl,triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any otherradicals substituted, especially mono- or di-substituted, by e.g. alkyl,nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinylrepresents preferably 2-, 3- or 4-quinolinyl. Isoquinolinyl representspreferably 1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranylrepresents preferably 3-benzopyranyl or 3-benzothiopyranyl,respectively. Thiazolyl represents preferably 2- or 4-thiazolyl, andmost preferred, 4-thiazolyl. Triazolyl is preferably 1-, 2- or5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl,thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl,thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl,benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted,especially mono- or di-substituted.

Similarly, substituents for the aryl and heteroaryl groups are variedand are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′,—NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, andperfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system; and where R′, R″ and R′″are independently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

As used herein, the term “visualization” refers to methods of obtaininggraphic images of tissue by any means, including illumination withnear-infrared radiation.

The term “near-infrared radiation” or “near IR radiation” refers tooptical radiation with a wavelength in the range of about 700 nm to 1400nm. References herein to the optionally plural term “wavelength(s)”indicates that the radiation may be a single wavelength or a spectrum ofradiation having differing wavelengths.

As used herein, the term “patient” denotes a mammal, such as a rodent, afeline, a canine, and a primate; most preferably said patient is ahuman.

The term “tissue” as used herein includes, but is not limited to,allogenic or xenogenic bone, neural tissue, fibrous connective tissueincluding tendons and ligaments, cartilage, dura, fascia, pericardia,muscle, heart valves, veins and arteries and other vessels, dermis,adipose tissue, glandular tissue, prostate tissue, kidney tissue, braintissue, renal tissue, bladder tissue, lung tissue, breast tissue,pancreatic tissue, vascular tissue, tumor tissue, cancerous tissue, orprostate tumor tissue.

As used herein, the term “sterile” refers to a system or components of asystem free of infectious agents including bacteria, viruses, andbioactive RNA or DNA.

As used herein, the term “non-toxic” refers to the non-occurrence ofdetrimental effects when administered to a vertebrate as a result ofusing a pharmaceutical composition at levels effective for visualizationof tissue under illumination with near-infrared radiation (therapeuticlevels).

The term “unit dosage form” as used herein encompasses any measuredamount that can suitably be used for administering a pharmaceuticalcomposition to a patient. Preferably, the unit dosage form is between0.01 and 8 mg/kg, or 0.01 and 5 mg/kg, or 0.01 and 1 mg/kg. Suitabledosage ranges also include 0.01, 0.05, 0.10, 0.20, 0.30, 0.35, 0.40,0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.90, 1, 2, 4, 6, or 8mg/kg. As recognized by those skilled in the art, when another form(e.g., another salt the pharmaceutical composition) is used in theformulation, the weight can be adjusted to provide an equivalent amountof the pharmaceutical composition.

The term “oral dosage form” as used herein refers to its normal meaningin the art (i.e., a pharmaceutical composition in the form of a tablet,capsule, caplet, gelcap, geltab, pill and the like).

The term “injectable dosage form” as used herein refers to its normalmeaning in the art (i.e., refer to a pharmaceutical composition in theform of solutions, suspensions, and emulsions, for example, water orwater/propylene glycol solutions.)

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a patient and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors and colors, and the like. One of skill in the art will recognizethat other pharmaceutical excipients are useful in the presentinvention. Pharmaceutically acceptable carriers include but not limitedto any adjuvants, excipients, glidants, sweeteners, diluents,preservatives, dyes/colorants, flavoring agents, surfactants, wettingagents, dispersing agents, suspending agents, stabilizing agents,isotonic agents, solvents or emulsors.

As used herein, the term “machine detectable identifier” includesidentifiers visible or detectable by machines including medical devices.In some instances, the medical device is a tele-surgical system. Machinedetectable identifiers may facilitate the access or utilization ofinformation that is directly encoded in the machine detectableidentifier, or stored elsewhere. Examples of machine detectibleidentifiers include microchips, radio frequency identification (RFID)tags, barcodes (e.g., 1-dimensional or 2-dimensional barcode), datamatrices, quick-response (QR) codes, and holograms. One of skill in theart will recognize that other machine detectible identifiers are usefulin the present invention.

III. Compositions

A. Compounds

The compounds of the present invention comprise fusion compounds offormulas I, II, and III which absorb light at wavelengths in thenear-infrared regions of the electromagnetic spectrum. In someembodiments, the present invention provides a composition comprising acompound having the formula:

wherein,

-   -   R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are each independently hydrogen,        C₁₋₄ alkyl, substituted C₁₋₄ alkyl, or —CO₂T;    -   X is a single bond, —O—, or —S—;    -   subscripts a, b, c, d, e, f, g, and h are each independently an        integer from 1 to 6, and    -   T is each independently a metal ion, H, or a negative charge; Z        is each independently H, C₁₋₆ substituted or unsubstituted        alkyl, C₆₋₁₂ substituted or unsubstituted aryl, or a C₆₋₁₂        substituted or unsubstituted heteroaryl; and

-   wherein, the composition is adapted for visualization of tissue    under illumination with near-infrared radiation.

In some other embodiments, the composition has the formula:

In some other embodiments, the composition has the formula:

The compounds of the present invention may exist as salts. The presentinvention includes such salts. Examples of applicable salt forms includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates,(−)-tartrates or mixtures thereof including racemic mixtures,succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in art.Also included are base addition salts such as sodium, potassium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When compounds of the present invention contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from organic acids like acetic, propionic,isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like. Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Other salts include acid or base salts of the compounds used in themethods of the present invention. Illustrative examples ofpharmaceutically acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, and quaternary ammonium (methyl iodide, ethyl iodide, and thelike) salts. It is understood that the pharmaceutically acceptable saltsare non-toxic. Additional information on suitable pharmaceuticallyacceptable salts can be found in Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., 1985, which isincorporated herein by reference.

Pharmaceutically acceptable salts include salts of the active compoundswhich are prepared with relatively non-toxic acids or bases, dependingon the particular substituents found on the compounds described herein.When compounds of the present invention contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredbase, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable base addition salts include sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively non-toxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, for example, Bergeet al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977,66, 1-19). Certain specific compounds of the present invention containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

In some embodiments, the present invention provides a pharmaceuticalcomposition including a pharmaceutically acceptable excipient.

B. Synthesis

The compounds described in the above embodiments may be made usingprocedures known in the art. In general, fusion compounds of the presentinvention can be synthesized by attaching near IR, closed chain,sulfo-cyanine dyes to prostate specific membrane antigen ligands via alinkage. The materials used can be determined by the desired structure,and the type of linkage used.

For example, the prostate specific membrane antigen ligands used in thecompositions of the present invention can be synthesized as described inPCT application WO 2010/108125 and is incorporated herein in itsentirety. Compounds can assembled by reactions between differentcomponents, to form linkages such as ureas (—NRC(O)NR—), thioureas(—NRC(S)NR—), amides (—C(O)NR— or —NRC(O)—), or esters (—C(O)O— or—OC(O)—). Urea linkages can be readily prepared by reaction between anamine and an isocyanate, or between an amine and an activatedcarbonamide (—NRC(O)—). Thioureas can be readily prepared from reactionof an amine with an isothiocyanate. Amides (—C(O)NR— or —NRC(O)—) can bereadily prepared by reactions between amines and activated carboxylicacids or esters, such as an acyl halide or Nhydroxysuccinimide ester.Carboxylic acids may also be activated in situ, for example, with acoupling reagent, such as a carbodiimide, or carbonyldiimidazole (CDI).Esters may be formed by reaction between alcohols and activatedcarboxylic acids. Triazoles are readily prepared by reaction between anazide and an alkyne, optionally in the presence of a copper (Cu)catalyst.

Prostate specific membrane antigen ligands can also be prepared bysequentially adding components to a preformed urea, such as thelysine-urea-glutamate compounds described in Banerjee et al. (J. Med.Chem. vol. 51, pp. 4504-4517, 2008). Other urea-based compounds may alsobe used as building blocks.

Exemplary syntheses of the near IR, closed chain, sulfo-cyanine dyesused in the compositions of the present invention are described in U.S.Pat. Nos. 6,887,854 and 6,159,657 and are incorporated herein in theirentirety (FIG. 1). Additionally, some IR, closed chain, sulfo-cyaninedyes of the present invention are commercially available, includingDyLight 800 (ThermoFisher).

As mentioned above, fusion compositions of the present invention can besynthesized via attachment of near IR, closed chain, sulfo-cyanine dyesto prostate specific membrane antigen ligands by reacting a reactiveamine on the ligand with a near IR dye (FIG. 2). A wide variety of nearIR dyes are known in the art, with activated functional groups forreacting with amines.

C. Formulation

The compositions of the present invention can be prepared in a widevariety of oral, parenteral and topical dosage forms. Oral preparationsinclude tablets, pills, powder, dragees, capsules, liquids, lozenges,cachets, gels, syrups, slurries, suspensions, etc., suitable foringestion by the patient. The compositions of the present invention canalso be administered by injection, that is, intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally. Also, the compositions described herein can beadministered by inhalation, for example, intranasally. Additionally, thecompositions of the present invention can be administered transdermally.The compositions of this invention can also be administered byintraocular, insufflation, powders, and aerosol formulations (forexamples of steroid inhalants, see Rohatagi, J. Clin. Pharmacol.35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111,1995). Accordingly, the present invention also provides pharmaceuticalcompositions including a pharmaceutically acceptable carrier orexcipient.

For preparing pharmaceutical compositions from the present invention,pharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. A solid carrier can beone or more substances, which may also act as diluents, flavoringagents, binders, preservatives, tablet disintegrating agents, or anencapsulating material. Details on techniques for formulation andadministration are well described in the scientific and patentliterature, see, e.g., the latest edition of Remington's PharmaceuticalSciences, Maack Publishing Co, Easton Pa. (“Remington's”).

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 5% or 10% to70% of the compounds of the present invention.

Suitable solid excipients include, but are not limited to, magnesiumcarbonate; magnesium stearate; talc; pectin; dextrin; starch;tragacanth; a low melting wax; cocoa butter; carbohydrates; sugarsincluding, but not limited to, lactose, sucrose, mannitol, or sorbitol,starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; aswell as proteins including, but not limited to, gelatin and collagen. Ifdesired, disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical compositions of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol. Push-fit capsules can contain thecompositions of the present invention mixed with a filler or binderssuch as lactose or starches, lubricants such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, thecompositions of the present invention may be dissolved or suspended insuitable liquids, such as fatty oils, liquid paraffin, or liquidpolyethylene glycol with or without stabilizers.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe compositions of the present invention in water and adding suitablecolorants, flavors, stabilizers, and thickening agents as desired.Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partialester derived from a fatty acid and a hexitol (e.g., polyoxyethylenesorbitol mono-oleate), or a condensation product of ethylene oxide witha partial ester derived from fatty acid and a hexitol anhydride (e.g.,polyoxyethylene sorbitan mono-oleate). The aqueous suspension can alsocontain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose, aspartame orsaccharin. Formulations can be adjusted for osmolarity.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Oil suspensions can be formulated by suspending the compositions of thepresent invention in a vegetable oil, such as arachis oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin;or a mixture of these. The oil suspensions can contain a thickeningagent, such as beeswax, hard paraffin or cetyl alcohol. Sweeteningagents can be added to provide a palatable oral preparation, such asglycerol, sorbitol or sucrose. These formulations can be preserved bythe addition of an antioxidant such as ascorbic acid. As an example ofan injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther.281:93-102, 1997. The pharmaceutical formulations of the invention canalso be in the form of oil-in-water emulsions. The oily phase can be avegetable oil or a mineral oil, described above, or a mixture of these.Suitable emulsifying agents include naturally-occurring gums, such asgum acacia and gum tragacanth, naturally occurring phosphatides, such assoybean lecithin, esters or partial esters derived from fatty acids andhexitol anhydrides, such as sorbitan mono-oleate, and condensationproducts of these partial esters with ethylene oxide, such aspolyoxyethylene sorbitan mono-oleate. The emulsion can also containsweetening agents and flavoring agents, as in the formulation of syrupsand elixirs. Such formulations can also contain a demulcent, apreservative, or a coloring agent.

The compositions of the present invention can also be delivered asmicrospheres for slow release in the body. For example, microspheres canbe formulated for administration via intradermal injection ofdrug-containing microspheres, which slowly release subcutaneously (seeRao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable andinjectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863,1995); or, as microspheres for oral administration (see, e.g., Eyles, J.Pharm. Pharmacol. 49:669-674, 1997). Both transdermal and intradermalroutes afford constant delivery for weeks or months.

In another embodiment, the compositions of the present invention can beformulated for parenteral administration, such as intravenous (IV)administration or administration into a body cavity or lumen of anorgan. The formulations for administration will commonly comprise asolution of the compositions of the present invention dissolved in apharmaceutically acceptable carrier. Among the acceptable vehicles andsolvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils canconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid can likewisebe used in the preparation of injectables. These solutions are sterileand generally free of undesirable matter. These formulations may besterilized by conventional, well-known techniques including radiation,chemical, heat/pressure, and filtration sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of the compositions ofthe present invention in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight, andthe like, in accordance with the particular mode of administrationselected and the patient's needs. For IV administration, the formulationcan be a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension can be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents. The sterile injectable preparation canalso be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

In another embodiment, the formulations of the compositions of thepresent invention can be delivered by the use of liposomes which fusewith the cellular membrane or are endocytosed, i.e., by employingligands attached to the liposome, or attached directly to theoligonucleotide, that bind to surface membrane protein receptors of thecell resulting in endocytosis. By using liposomes, particularly wherethe liposome surface carries ligands specific for target cells, or areotherwise preferentially directed to a specific organ, one can focus thedelivery of the compositions of the present invention into the targetcells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306,1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J.Hosp. Pharm. 46:1576-1587, 1989).

Lipid-based drug delivery systems include lipid solutions, lipidemulsions, lipid dispersions, self-emulsifying drug delivery systems(SEDDS) and self-microemulsifying drug delivery systems (SMEDDS). Inparticular, SEDDS and SMEDDS are isotropic mixtures of lipids,surfactants and co-surfactants that can disperse spontaneously inaqueous media and form fine emulsions (SEDDS) or microemulsions(SMEDDS). Lipids useful in the formulations of the present inventioninclude any natural or synthetic lipids including, but not limited to,sesame seed oil, olive oil, castor oil, peanut oil, fatty acid esters,glycerol esters, Labrafil®, Labrasol®, Cremophor®, Solutol®, Tween®,Capryol®, Capmul®, Captex®, and Peceol®.

In some embodiments the compositions of the present invention aresterile and generally free of undesirable matter. The compounds andcompositions may be sterilized by conventional, well known techniquesincluding heat/pressure, gas plasma, steam, radiation, chemical, andfiltration sterilization techniques.

For example, terminal heat sterilization can be used to destroy allviable microorganisms within the final formulation. An autoclave iscommonly used to accomplish terminal heat-sterilization of drug productsin their final packaging. Typical autoclave cycles in the pharmaceuticalindustry to achieve terminal sterilization of the final product are 121°C. for 15 minutes. The compositions of the present invention can beautoclaved at a temperature ranging from 115 to 130° C. for a period oftime ranging from 5 to 40 minutes with acceptable stability. Autoclavingis preferably carried out in the temperature range of 119 to 122° C. fora period of time ranging from 10 to 36 minutes.

The compositions can also be sterilized by dry heat as described byKarlsson, et al., in U.S. Pat. No. 6,392,036, which discloses a methodfor the dry heat sterilization that can be used for drug formulations.The compositions can also be sterilized as described in WO 02/41925 toBreath Limited, which discloses a rapid method, similar topasteurization, for the sterilization of compositions. This methodentails pumping the composition to be sterilized through stainless steelpipes and rapidly raising the temperature of the composition to about130-145° C. for about 2-20 seconds, subsequently followed by rapidcooling in seconds to ambient conditions.

The compositions can also be sterilized by irradiation as described byIllum and Moeller in Arch. Pharm. Chemi. Sci., Ed. 2, 1974, pp.167-174). The compositions can also be sterilized by UV, x-rays, gammarays, e beam radiation, flaming, baking, and chemical sterilization.

Alternatively, sterile pharmaceutical compositions according to thepresent invention may be prepared using aseptic processing techniques.Aseptic filling is ordinarily used to prepare drug products that willnot withstand heat sterilization, but in which all of the ingredientsare sterile. Sterility is maintained by using sterile materials and acontrolled working environment. All containers and apparatus aresterilized, preferably by heat sterilization, prior to filling. Thecontainer (e.g., vial, ampoule, infusion bag, bottle, or syringe) arethen filled under aseptic conditions.

In some embodiments, the compounds and compositions of the presentinvention are non-toxic and generally free of detrimental effects whenadministered to a vertebrate at levels effective for visualization oftissue under illumination with near-infrared radiation. Toxicity of thecompounds and compositions of the present invention can be assessed bymeasuring their effects on a target (organism, organ, tissue or cell).Because individual targets typically have different levels of responseto the same dose of a compound, a population-level measure of toxicityis often used which relates the probabilities of an outcome for a givenindividual in a population. Toxicology of compounds can be determined byconventional, well-known techniques including in vitro (outside of aliving organism) and in vivo (inside of a living organism) studies.

For example, determination of metabolic stability is commonly examinedwhen assessing the toxicity of a compound as it is one of several majordeterminates in defining the oral bioavailability and systemic clearanceof a compound. After a compound is administered orally, it firstencounters metabolic enzymes in the gastrointestinal lumen as well as inthe intestinal epithelium. After it is absorbed into the bloodstreamthrough the intestinal epithelium, it is first delivered to the livervia the portal vein. A compound can be effectively cleared by intestinalor hepatic metabolism before it reaches systemic circulation, a processknown as first pass metabolism. The stability of a compound towardsmetabolism within the liver as well as extrahepatic tissues willultimately determine the concentration of the compound found in thesystemic circulation and affect its half-life and residence time withinthe body. Cytochrome P450 (CYP) enzymes are found primarily in the liverbut also in the intestinal wall, lungs, kidneys and other extrahepaticorgans and are the major enzymes involved in compound metabolism. Manycompounds undergo deactivation by CYPs, either directly or byfacilitated excretion from the body. Also, many compounds arebioactivated by CYPs to form their active compounds. Thus, determiningthe reactivity of a compound to CYP enzymes is commonly used to assessmetabolic stability of a compound.

The Ames reverse mutation Assay is another common toxicology assay forassessing the toxicity of a compound. The Ames Assay, utilizes severaldifferent tester strains, each with a distinct mutation in one of thegenes comprising the histidine (his) biosynthetic operon (Ames, B. N.,et al., (1975) Mutation Res. 31:347-363). The detection of revertant(i.e., mutant) bacteria in test samples that are capable of growth inthe absence of histidine indicates that the compound under evaluation ischaracterized by genotoxic (i.e. mutagenic) activity. The Ames Assay iscapable of detecting several different types of mutations (geneticdamage) that may occur in one or more of the tester strains. Thepractice of using an in vitro bacterial assay to evaluate the genotoxicactivity of drug candidates is based on the prediction that a substancethat is mutagenic in a bacterium is likely to be carcinogenic inlaboratory animals, and by extension may be carcinogenic or mutagenic tohumans.

In addition, the human ether-a-go-go related gene (hERG) assay can beused to evaluate the potential cardiotoxicity of a compound.Cardiotoxicity can arise when the QT interval is prolonged leading to anelevated risk of life-threatening arrhythmias. The QT interval is ameasure of the time between the start of the Q wave and the end of the Twave in the heart's electrical cycle. The QT interval representselectrical depolarization and repolarization of the ventricles. Alengthened QT interval has most commonly been associated with loss ofcurrent through hERG potassium ion channels due to direct block of theion channel by drugs or by inhibition of the plasma membrane expressionof the channel protein (Su et al. J. Biomol Screen 2011, 16, 101-111).Thus, an in vitro hERG screening assay can be used to detect disruptionor inhibition of the hERG membrane trafficking function and assesspotential cardiotoxicity of a compound.

Other methods of assessing the toxicity of compounds include in vivostudies which administer relatively large doses of a test compound to agroup of animals to determine the level which is lethal to a percentageof the population (mean lethal dose LD₅₀ or mean lethal concentrationLC₅₀). Toxicity of a compound can also be assessed in vivo by examiningwhether a compound produces statistically significant negative effectson cardiac, blood pressure, central nervous system (CNS), body weight,food intake, gross or microscopic pathology, clinical pathology, orrespiratory measures in an animal.

For example, in a set of in vitro studies evaluating the metabolicstability of a composition of formula III, it was shown that thecompound appears to be stable in rat, dog and human plasma, does notappear to be broken down into metabolites, and does not show anysignificant reactivity to 9 major CYP liver enzymes. Additionally, thecomposition of formula III does not show any mutagenicity at any of thetested doses in the Ames reverse mutation assay, a widely used in vitromethod that determines the ability of a chemical to cause mutations inDNA. Further safety pharmacology was assessed in vitro using the humanether-a-go-go related gene (hERG) assay for determining possiblecardio-toxic effects. Studies utilizing this assay determined that thecomposition of formula III shows only a small (11%) inhibition of hERGfunction at the highest tested concentration (30 times higher thanlevels effective for visualization), indicating even at thisconcentration the composition of formula III is unlikely to translateinto any clinically threatening physiological cardiac changes.

Furthermore, toxicology of the composition of formula III was alsoinvestigated in non-clinical in vivo studies in both rats and dogs. Astudy evaluating the effects of the composition of formula III oncardiac and respiratory safety in radio-telemetry monitored dogs showedno statistically significant negative effects on cardiac, blood pressureor respiratory measures at doses up to 80 times higher than thoserequired for visualization. Additional safety pharmacology performedover 28 days in rats indicated doses as high as 160 times visualizationlevels per day showed no biologically meaningful effects on the centralnervous system (CNS). Finally, a series of toxicology studies in dogsshowed doses as high as 100 times visualization levels showed nosignificant effects on body weight, food intake, gross or microscopicpathology as well as clinical pathology (clinical serum chemistry,hematology, coagulation and urinalysis). No mortality was observed atany tested dose of the composition of formula III in rat, dog or piganimal models.

In some embodiments, the compositions of the present invention can belyophilized in a sterile container for convenient dry storage andtransport. A ready-to-use preparation can subsequently be made byreconstituting the lyophilized compositions with sterile water. Theterms “lyophilization,” “lyophilized,” and “freeze-dried” refer to aprocess by which the material to be dried is first frozen and then theice or frozen solvent is removed by sublimation in a vacuum environment.An excipient may be included in pre-lyophilized formulations to enhancestability of the lyophilized product upon storage.

In some embodiments, the composition can be contained within a sterilecontainer, where the container has a machine detectable identifier whichis readable by a medical device. Examples of machine detectibleidentifiers include microchips, radio frequency identification (RFID)tags, barcodes (e.g., 1-dimensional or 2-dimensional barcode), datamatrices, quick-response (QR) codes, and holograms. One of skill in theart will recognize that other machine detectible identifiers are usefulin the present invention.

In some cases, the machine detectable identifier can include amicrochip, an integrated circuit (IC) chip, or an electronic signal froma microchip that is detectable and/or readable by a computer system thatis in communication with the medical device. In some cases, the machinedetectable identifier includes a radio frequency identification (RFID)tag. RFID tags are sometimes called as transponders. RFID tags generallyare devices formed of an IC chip, an antenna, an adhesive material, andare used for transmitting or receiving predetermined data with anexternal reader or interrogator. RFID tags can transmit or receive datawith a reader by using a contactless method. According to the amplitudeof a used frequency, inductive coupling, backscattering, and surfaceacoustic wave (SAW) may be used. Using electromagnetic waves, data maybe transmitted or received to or from a reader by using a full duplexmethod, a half duplex (HDX) method, or a sequential (SEQ) method.

In some cases, the machine detectable identifier can include a barcode.Barcodes include any machine-readable format, including one-dimensionaland two-dimensional formats. One-dimensional formats include, forexample, Universal Product Code (UPC) and Reduced Space Symbology (RSS).Two-dimensional formats, or machine-readable matrices, include forexample, Quick Response (QR) Code and Data Matrix.

In some cases, the medical device can be configured to detect themachine detectable identifier. In one example, the medical device is atele-surgical system that includes a special imaging mode (e.g., afluorescence imaging mode) for use with dyes such as those described inthis disclosure. One example of a tele-surgical system that includes afluorescence imaging mode is described in U.S. Pat. No. 8,169,468,entitled “Augmented Stereoscopic Visualization for a Surgical Robot,”which is hereby incorporated in its entirety herein. In some cases,medical devices can incorporate an imaging device that can scan, read,view, or otherwise detect a machine detectable identifier that isdisplayed to the imaging device. In one aspect, the medical device willpermit a user to access the fluorescence imaging mode of the medicaldevice only if the medical device detects the presence of a knownmachine detectable identifier that corresponds to a dye identified asbeing compatible for use with the medical device. In contrast, if themedical device does not detect a known machine detectable identifier,the medical device will not permit a user to access the fluorescenceimaging mode and associated functionality. Imaging devices can includeoptical scanners, barcode readers, cameras, and imaging devicescontained within a tele-surgical system such as an endoscope.Information associated with the machine detectable identifier can thenbe retrieved by the medical device using an imaging device. Upondetection of the identifier, an automatic process may be launched tocause a predetermined action to occur, or certain data to be retrievedor accessed. The information encoded onto the machine detectableidentifier may include instructions for triggering an action, such asadministering a composition of the present invention to a patient. Insome embodiments, the machine detectable identifier includes unencryptede-pedigree information in the desired format. The e-pedigree informationcan include, for example, lot, potency, expiration, national drug code,electronic product code, manufacturer, distributor, wholesaler, pharmacyand/or a unique identifier of the salable unit.

In some embodiments, the sterile container having a machine detectableidentifier includes a fluid outlet configured to mate with the medicaldevice. In some cases, the fluid outlet of the machine detectableidentifier is mechanically affixed to the medical device.

D. Administration

The compounds and compositions of the present invention can be deliveredby any suitable means, including oral, parenteral and topical methods.

The compounds and compositions of the present invention can be deliveredby any suitable means, including oral, parenteral and topical methods.Transdermal administration methods, by a topical route, can beformulated as applicator sticks, solutions, suspensions, emulsions,gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the compounds and compositions of the presentinvention. The unit dosage form can be a packaged preparation, thepackage containing discrete quantities of preparation, such as packetedtablets, capsules, and powders in vials or ampoules. Also, the unitdosage form can be a capsule, tablet, cachet, or lozenge itself, or itcan be the appropriate number of any of these in packaged form.

In some embodiments, co-administration can be accomplished byco-formulation, i.e., preparing a single pharmaceutical compositionincluding the compounds and compositions of the present invention andany other agent. Alternatively, the various components can be formulatedseparately.

The compositions of the present invention, and any other agents, can bepresent in any suitable amount, and can depend on various factorsincluding, but not limited to, weight and age of the patient, state ofthe disease, etc. Suitable dosage ranges include from about 0.01 and 8mg/kg, or about 0.01 and 5 mg/kg, or about 0.01 and 1 mg/kg. Suitabledosage ranges also include 0.01, 0.05, 0.10, 0.20, 0.30, 0.35, 0.40,0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.90, 1, 2, 4, 6, or 8mg/kg.

The composition can also contain other compatible compositions. Thecompositions described herein can be used in combination with oneanother, with other active compositions known to be useful forvisualization of tissue under illumination with near-infrared radiation,or with compositions that may not be effective alone, but may contributeto the efficacy of the active composition.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding equivalents of thefeatures shown and described, or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention claimed. Moreover, any one or more features of any embodimentof the invention may be combined with any one or more other features ofany other embodiment of the invention, without departing from the scopeof the invention. All publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

IV. Methods for Visualization of Tissue

The present invention generally provides novel, compositions and methodsfor visualizing tissue under illumination with fusion compoundscomprising near-infrared radiation using near IR, closed chain,sulfo-cyanine dyes and prostate specific membrane antigen ligands.

In some embodiments, the present invention provides a method forvisualization of tissue expressing PSMA, the method comprising,administering to a patient a composition of a compound having theformula:

wherein,

-   -   R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are each independently hydrogen,        C₁₋₄ alkyl, substituted C₁₋₄ alkyl, or —CO₂T;    -   X is a single bond, —O—, or —S—;    -   subscripts a, b, c, d, e, f, g, and h are each independently an        integer from 1 to 6;    -   T is each independently a metal ion, H, or a negative charge; Z        is each independently H, C₁₋₆ substituted or unsubstituted        alkyl, C₆₋₁₂ substituted or unsubstituted aryl, or a C₆₋₁₂        substituted or unsubstituted heteroaryl; and where the compound        is administered in an amount sufficient for imaging tissue under        illumination with near-infrared radiation;        imaging the tissue under illumination with near-infrared        radiation; and obtaining at least one image of tissue from the        patient using the composition.

In some embodiments, the method administers to a patient apharmaceutical composition comprising a unit dosage form of a compoundof formula I wherein, the composition is sterile, non-toxic, and adaptedfor administration to a patient; and wherein, the unit dosage form ofthe compound delivers to the patient an amount between 0.01 and 8 mg/kg.In some cases, the method further comprises obtaining the image duringadministration, after administration, or both during and afteradministration of the composition. In some cases, the method furthercomprises intravenously injecting a composition of formula I into apatient. In some cases, the composition is injected into a circulatorysystem.

In some embodiments, the present invention provides a use of thecomposition adapted for administration to a patient to obtainvisualization of tissue expressing PSMA under illumination withnear-infrared radiation wherein the unit dosage form delivers to thepatient an amount is between 0.01 and 8 mg/kg. In some cases, the use isadapted for administration to a human patient to obtain visualization ofhuman tissue under illumination with near-infrared radiation wherein theunit dosage form delivers to the patient an amount between 0.01 and 8mg/kg.

In some embodiments, the method further comprises visualizing a patientarea on which surgery is or will be performed, or for viewing a patientarea otherwise being examined by a medical professional. In some cases,the method further comprises performing a surgical procedure on thepatient areas based on the visualization of the surgical area. In somecases, the method further comprises viewing a patient area on which anophthalmic, arthroscopic, laparoscopic, cardiothoracic, muscular, orneurological procedure is or will be performed. In some cases, themethod further comprises obtaining ex vivo images of at least a portionof the patient.

In some embodiments, improvement in visualization of tissue can beachieved through the use of dyes capable of targeted visualization oftissue, including by fusion compositions of near IR, closed chain,sulfo-cyanine dyes and prostate specific membrane antigen (PSMA)ligands. The PSMA, while expressed in prostate tumor epithelium, is alsoexpressed in the neovasculature of many solid tumors (Chang et al.,Cancer Res., vol. 59, pp. 3192-3198, 1999; Chang et al., Clin. CancerRes., vol. 5, pp. 2674-2681, 1999; Gong et al., Cancer Metastasis Rev.,vol. 18, pp. 483-490, 1999; Chang et al., Mol. Urol., vol. 3, pp.313-320, 1999; Baccala et al., Urology, vol. 70, pp. 385-390, 2007;Chang et al., Urology, vol. 57, pp. 801-805, 2001 Milowsky et al., JClin. Oncol., vol. 25, pp. 540-547, 2007). As a result, PSMA ligandshave been used to identify renal, bladder, lung, breast, colorectal, andpancreatic tumors (Milowsky et al., J Clin. Oncol., vol. 25, pp.540-547, 2007). PSMA ligands have proved to be an effective vasculartargeting agent in human subjects. Other reports have further studiedPSMA expression in certain tumor types. For example, Baccala et al.noted that clear cell renal cell carcinoma expresses significantly morePSMA in its neovasculature than does the papillary variety (Baccala etal., Urology, vol. 70, pp. 385-390, 2007). Furthermore, angiomyolipoma,a benign renal lesion, has been found not to express PSMA. As an enzymewith an extracellular active site, PSMA represents an excellent targetfor imaging and therapy directed toward solid tumor neovasculature inaddition to prostate cancer itself. PSMA-based agents can report on thepresence of this marker, which is increasingly recognized as animportant prognostic determinate in PCa (Murphy et al., Urology, vol.51, pp. 89-97, 1998). It is also the target for a variety of new PCatherapies (Gal sky et al., J Clin Oneal, vol. 26, pp. 214 7-2154, 2008).

Accordingly, compositions of the present invention comprising fusioncompounds of near IR, closed chain, sulfo-cyanine dyes and PSMA ligandscan be used to visualize a variety of tumor tissues. In some cases, thetumor tissue being visualized is prostate tissue, kidney tissue, braintissue, renal tissue, bladder tissue, lung tissue, breast tissue,pancreatic tissue, vascular tissue, tumor tissue, cancerous tissue, orprostate tumor tissue. In some cases, the tissue being visualized iscancerous tissue. In some cases, the tissue being visualized is prostatetissue. In some cases, the tissue being visualized is prostate tumortissue.

In some cases, the method further comprises the use of compositions ofthe present invention in combination with the da Vinci Surgical System'sFirefly fluorescence, in providing an augmented view that enhances thedifficult-to-visualize tissue to increase surgical efficacy, reduce theinjury rate, and possibly speed up surgery by providing the surgeon withconfidence and security that he/she is removing all intended tissue. Forexample, positive margin rates during prostate cancer surgery can be ashigh as 30%, meaning that cancerous tissue is left within a man despiteattempts to remove it all. Such patients frequently require subsequenttherapy, including but not limited to external beam radiation therapy,brachytherapy, ablative and hormonal therapy. In addition, rates oferectile dysfunction and incontinence, resulting from nerve injuryduring prostate surgery can approach 50% and 20% respectively.Compositions of the present invention comprising fusion compounds ofnear IR, closed chain, sulfo-cyanine dyes and PSMA ligands can also beused to increase contrast between prostatic and nerve tissue, helping toavoid resulting injury to nerves and sphincter tissue. Accordingly, theuse of compositions of the present invention in combination with the daVinci Surgical System's Firefly fluorescence, provide an augmented viewthat enhances the difficult-to-visualize tissue to reduce positivesurgical margin rates, reduce adverse side effects, and possibly speedup surgery.

V. Examples Example 1. Visualization of Tumor Tissue Under Illumination

Four similar tumor tissues were treated with four different fusioncompounds comprising different dyes and targeting ligands. The fourfusion compounds include DyLight800 and PSMA-targeting ligand,IRDye800CW and a PSMA-targeting ligand, ZW800 and PSMA-targeting ligand,and ICG and a PSMA-targeting ligand. The treated tumor tissue in each ofthe four images was then exposed to narrow band, near IR excitationlight in order to produce FIGS. 1A-1D. The narrow band, near IRexcitation light included light wavelengths expected to correspond tothe excitation maxima wavelength for each of the fluorophores associatedwith each of the different compounds.

As shown in FIG. 2, the fusion compound comprising DyLight800 and aPSMA-targeting ligand exhibited significantly more fluorescence. Thesignificant differences in fluorescence may be due to at least one ofthe following reasons: (1) Attaching ZW800, IRDye800CW, or ICG toPSMA-targeting ligand adversely affected the binding affinity of thePSMA-targeting ligand to PSMA binding sites on tumor tissue; and (2)Attaching ZW800, IRDye800CW, or ICG to PSMA-targeting ligand adverselyaffected the fluorescent properties of the ZW800, IRDye800CW, or ICGfluorescent moiety. In contrast, conjugating DyLight800 toPSMA-targeting ligand had neither of these adverse effects.

Example 2. Visualization of Prostatic Tissue Under Illumination

20 mg of a compound of formula III in 10 ml of sterile water can beadministered intravenously to patients. Laparoscopic ports can then beplaced and the da Vinci Surgical System connected to the ports. Theendoscope of the system can then be directed at the prostate of thepatient, and laser excitation at approximately 800 nm can be used toexcite the composition of formula III within the prostate. A smallamount of blue and green light can also be emitted in order to allowvisualization of the background anatomy. Approximately 2-24 hours afteradministration, visualization of the prostate and prostate tumor tissuecan be achieved as the composition of formula III has bound to PSMA.

1. A sterile, non-toxic pharmaceutical composition suitable foradministration to a patient, comprising: (a) a pharmaceuticallyacceptable excipient; and (b) a unit dosage form of a compound havingthe formula:

wherein: R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are each independently selectedfrom the group consisting of hydrogen, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl; X is selectedfrom the group consisting of a single bond, —O—, and —S—; subscripts a,b, c, d, e, f, g, and h are each independently an integer selected from1 to 6; each T is independently selected from the group consisting of ametal ion, H, and a negative charge; and each Z is independentlyselected from the group consisting of hydrogen, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branchedpentyl, n-hexyl, and branched hexyl, and wherein the unit dosage formdelivers to the patient between 0.01 and 8 mg/kg of the compound offormula (I); and wherein the pharmaceutical composition is adapted forvisualization of tissue expressing PSMA under illumination atwavelengths ranging from 700 to 1400 nm.
 2. The pharmaceuticalcomposition of claim 1, wherein the unit dosage form delivers to thepatient 0.01, 0.05, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55,0.60, 0.65, 0.70, 0.75, 0.80, 0.90, 1, 2, 4, 6, or 8 mg/kg of thecompound of formula (I).
 3. The pharmaceutical composition of claim 1,wherein the composition is: (a) dry and a single dose form; (b)lyophilized in a sterile container; or (c) contained within a sterilecontainer, wherein the container has a machine detectable identifierwhich is readable by a medical device.
 4. The pharmaceutical compositionof claim 1, wherein the composition is in an oral dosage form.
 5. Thepharmaceutical composition of claim 1, wherein the composition is in aninjectable dosage form.
 6. The pharmaceutical composition of claim 1,wherein the composition is in a dosage form for direct delivery to asurgical site.
 7. A sterile, non-toxic pharmaceutical compositionsuitable for administration to a patient, comprising: (a) apharmaceutically acceptable excipient; and (b) a unit dosage form of acompound having the formula:

wherein the unit dosage form delivers to the patient between 0.01 and 8mg/kg of the compound of formula (III); and wherein the pharmaceuticalcomposition is adapted for visualization of tissue expressing PSMA underillumination at wavelengths ranging from 700 to 1400 nm.
 8. Thepharmaceutical composition of claim 7, wherein the unit dosage formdelivers to the patient 0.01, 0.05, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45,0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.90, 1, 2, 4, 6, or 8 mg/kgof the compound of formula (III).
 9. The pharmaceutical composition ofclaim 7, wherein the composition is: (a) dry and a single dose form; (b)lyophilized in a sterile container; or (c) contained within a sterilecontainer, wherein the container has a machine detectable identifierwhich is readable by a medical device.
 10. The pharmaceuticalcomposition of claim 7, wherein the composition is in an oral dosageform.
 11. The pharmaceutical composition of claim 7, wherein thecomposition is in an injectable dosage form.
 12. The pharmaceuticalcomposition of claim 7, wherein the composition is in a dosage form fordirect delivery to a surgical site.
 13. A method for visualization oftissue expressing PSMA, the method comprising: (i) administering to apatient a sterile, non-toxic pharmaceutical composition comprising: (a)a pharmaceutically acceptable excipient; and (b) a unit dosage form of acompound having the formula:

wherein the unit dosage form delivers to the patient between 0.01 and 8mg/kg of the compound of formula (III); and wherein the pharmaceuticalcomposition is administered in an amount sufficient for imaging tissueunder illumination with near-infrared radiation; (ii) imaging the tissueunder illumination with near-infrared radiation ranging from 700 to 1400nm; and (iii) obtaining at least one image of the tissue from thepatient.
 14. The method of claim 13, wherein obtaining the image occursduring administration, after administration, or both during and afteradministration of the pharmaceutical composition.
 15. The method ofclaim 13, wherein the pharmaceutical composition is administered to thepatient by intravenous injection.
 16. The method of claim 15, whereinthe pharmaceutical composition is injected into a circulatory system.17. The method of claim 13, wherein the tissue is in a patient area onwhich surgery is or will be performed, or wherein the tissue is in apatient area being examined by a medical professional.
 18. The method ofclaim 17, further comprising: (a) viewing the patient area on which anophthalmic, arthroscopic, laparoscopic, cardiothoracic, muscular, orneuro procedure is or will be performed; and/or (b) performing asurgical procedure on the patient area based on the visualization of thetissue.
 19. The method of claim 18, further comprising obtaining ex vivoimages of at least a portion of the patient area.
 20. The method ofclaim 13, wherein the tissue being visualized is: (a) tumor tissue; (b)cancerous tissue; (c) prostate tissue; (d) prostate tumor tissue; or (e)nerve tissue.